Influenza T-cell immunization against diverse influenza A viruses

ABSTRACT

There is provided a T cell-based universal influenza vaccine including internal genes capable of preparing against infection by broad hetero-subtypic influenza viruses, and thereby preparing for unpredictable epidemic influenza. The present invention selected internal genes of the consensus sequence obtained from bird, pig, and human influenza isolates in order to develop the T cell-based universal flu vaccine. The T cell-based universal flu vaccine according to the present invention is characterized by including at least one CTL epitope, by containing a plurality of internal genes and using the entire sequence of the internal gene itself. The T cell-based universal flu vaccine can achieve broad defense in infection with hetero-subtypic influenza viruses.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to Korean PatentApplication No. 10-2012-0033317, filed on Mar. 30, 2012, in the KoreanIntellectual Property Office, the disclosure of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The following disclosure relates to a T cell-based universal influenzavaccine capable of preparing against infection by broad hetero-subtypicinfluenza viruses and thereby preparing for unpredictable epidemicinfluenza.

BACKGROUND

Since the 20^(th) century, influenza infectious diseases have claimed ahuge number of victims (Nat. Immunol 7,449-455, 2006). Although it hasbeen possible to predict variant viruses that will prevail in the seasonnext year due to the preparation for seasonal influenza by the GlobalInfluenza Surveillance Network (GISN), there still remains uncertaintyin that the predicted vaccine strains do not correspond to the epidemicstrains of the following year or recombinant vaccine strains areimpossible to mass-produce. The epidemic of the Fujian strain from 2003to 2004, a variant of vaccine strain A/Panama/2007/99, caused the annualaverage infant mortality rate to rapidly increase from 9 to 153 in thefollowing year (N Engl J Med 350,218-220, 2004). This may be arepresentative example in which the predicted vaccine candidate straindoes not match to the epidemic strain. Unfortunately, the known existingegg-based production process failed to overcome the above disadvantagesin the production of influenza vaccine.

The Spanish flu notoriously increased the mortality rate due tounprecedented extreme toxicity. In addition, appearance of highlypathogenic avian influenza (HPAI) H5N1, which has similar pathogenicityto the Spanish flu highlighted the fact that it is very difficult andimpossible to prepare for infectious diseases that will prevail in thefuture (Nature 437, 889-893, 2005). The Global Influenza SurveillanceNetwork (GISN) has conducted epidemiological survey of H5N1 since 2004,and regularly updated the vaccine candidates. Most vaccine manufacturershave developed H5N1 prepandemic vaccine according to the prediction ofGISN, and currently, this is the only measure.

The pandemic H1N1 and highly pathogenic avian influenza (HPAI) H5N1 in2009 caused concerns that highly pathogenic and highly infectiousrecombinant influenza reassortants might appear in the future.Eventually, only the universal influenza vaccine capable of preventinginfection by broad types of viruses would be the ultimate solution.There was a study of proposing a M2 protein as a promising target foruniversal influenza vaccine (Nat Med 5, 1157-1163, 1999). Since the M2protein exhibits a significant level of expression on a surface of thecell infected with virus, it can prevent disease or death due to highlypathogenic influenza viruses by using anti-M2 antibodies in cellmediated immunity for removing the cells infected with viruses, butthere are some limits therein.

In addition, the combinations of adjuvant agents, virus-like particles(VLP), or inactive seasonal influenza vaccine, for enhancingimmunogenicity of M2, are being investigated.

Recently, a highly conserved hemagglutinin (HA) stem region of theinfluenza virus is drawing attention, and a relatively conserved HA stemis receiving attention as an attractive target capable of neutralizingviruses broadly. Studies about new vaccines against different types ofinfluenza by using these among several groups are being conducted.However, anti-HA stem antibodies have relatively low neutralizationactivity similarly to the anti-M2 antibodies because they are not directneutralizing antibodies. Therefore, as long as there are not otherdefense cooperation means in an antibody-meditated cell immunity aimingon defense, the anti-HA stem antibodies are effective only when thechallenge dose is low. Various approaches for T cell-based vaccine havebeen attempted even though there were no commercially successful cases.Recent studies about HIV and HCV vaccines represented several promisingantigen delivery means for inducing broad and active T cell immunity atthe time of defense against highly variable viruses. According to theresults of study about historical events of the Asian influenza, whichwas conducted in Cleveland in the year 1957, it can be found that thepresence of cross-reactive memory T cells induced by the existinginfected viruses has a defense effect even when hetero-subtypicinfluenza newly prevail. T-cell response has been supposed as a crossdefense immune reaction against influenza infection in many clinicalresearches and animal studies including ferrets and monkeys. Theincrease in T-cell immunity due to re-infection by hetero-subtypicinfluenza viruses eventually showed that, the higher the T-cellimmunity, the more efficient the defense against highly pathogenicinfluenza infection. Many research groups have studied antigen deliverymethods by highly conserved internal proteins or epitope of influenzavirus, such as NP or M1, by using DNA, adenovirus, poxvirus, or liveattenuated virus.

Epstein researchers showed that recombinant adenovirus expressing NP andM2 had surprising efficacy in the defense against hetero-subtypicviruses when intranasal administration of vaccine was conducted and afatal dose of challenge was attempted. T-cell vaccine has an advantageof fast induction for defense immunity, and in particular, the defenseimmunity is induced in one week after immunization. Many studies haveproved that vaccinia virus has promising possibility as a T-cell vaccinevector.

PRIOR ART DOCUMENTS Non-Patent Documents

-   1. (Nat Immunol 7,449-455, 2006)-   2. (N Engl J Med 350,218-220, 2004)-   3. (Nature 437,889-893, 2005)-   4. (Nat Med 5, 1157-1163, 1999)

SUMMARY

An embodiment of the present invention is directed to providing a T-cellbased universal influenza vaccine containing internal genes allowingbroad defense against various hetero-subtypic variants of influenzaviruses.

The present invention provides a T-cell based universal influenzavaccine containing internal genes capable of defending broad types ofinfluenza variants in order to prepare for unpredictable epidemicinfluenza.

Further, the present invention provides a T-cell based universalinfluenza vaccine containing, as effective components, five kinds ofinternal genes, NP (SEQ ID NO: 1), PA (SEQ ID NO: 3), PB1 (SEQ ID NO:5), PB2 (SEQ ID NO: 7), and M1 (SEQ ID NO: 9), which have a consensussequence and are selected from the bird, pig, and human influenzaisolates. An aspect of the present invention provides a T-cell baseduniversal influenza vaccine containing, as effective components, NP (SEQID NO: 1) and PA (SEQ ID NO: 3) genes. Another aspect of the presentinvention provides a T-cell based universal influenza vaccinecontaining, as effective components, NP (SEQ ID NO: 1) and PA (SEQ IDNO: 3) genes, and further containing, as another effective component, atleast one selected from PB1 (SEQ ID NO: 5), PB2 (SEQ ID NO: 7), and M1(SEQ ID NO: 9) genes. Still another aspect of the present inventionprovides a T-cell based universal influenza vaccine containing all of NP(SEQ ID NO: 1), PA (SEQ ID NO: 3), PB1 (SEQ ID NO: 5), PB2 (SEQ ID NO:7), and M1 (SEQ ID NO: 9) genes.

In addition, in the T-cell based universal influenza vaccine:

a CTL epitope of the NP gene has a sequence consisting of:

(SEQ ID NO: 11)  1. MASQGTKRSYEQMET, (SEQ ID NO: 12) 2. GIGRFYIQMCTELKL, (SEQ ID NO: 13)  3. MVLSAFDERRN, (SEQ ID NO: 14) 4. YLEEHPSAGKDPKKTGGPIY, (SEQ ID NO: 15)  5. LYDKEEIRRIWRQANNG,(SEQ ID NO: 16)  6. ATYQRTRAL, (SEQ ID NO: 17)  7. YERMCNILKG,(SEQ ID NO: 18)  8. QVRESRNPGNAEIEDLIFLA, (SEQ ID NO: 19) 9. QLVWMACHSAAFEDLRVSSF, or (SEQ ID NO: 20) 10. QPTFSVQRNLPF;a CTL epitope of the PA gene has a sequence consisting of:

(SEQ ID NO: 21)  1. KIETNKFAAICTHLEVCFMYSDFHF, (SEQ ID NO: 22) 2. RTMAWTVVNSI, (SEQ ID NO: 23)  3. VEKPKFLPDLY, (SEQ ID NO: 24) 4. YYLEKANKIKSE, (SEQ ID NO: 25)  5. THIHIFSFTGEEMA, (SEQ ID NO: 26) 6. RGLWDSFRQSERGEETIEE, (SEQ ID NO: 27)  7. RSKFLLMDALKLSIE,(SEQ ID NO: 28)  8. HEGEGIPLYDAIKC, (SEQ ID NO: 29)  9. SQLKWALGENMA,(SEQ ID NO: 30) 10. EFNKACELTDSSWI, (SEQ ID NO: 31) 11. SRPMFLYVRTNGTSK,or (SEQ ID NO: 32) 12. AESRKLLLI;a CTL epitope of the PB1 gene has a sequence consisting of:

(SEQ ID NO: 33)  1. MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSE,(SEQ ID NO: 34)  2. MAFLEESHPGIFENS, (SEQ ID NO: 35) 3. VQQTRVDKLTQGRQTYDWTLNRNQPAATALANTIE, (SEQ ID NO: 36) 4. TKKMVTQRTIGKKK, (SEQ ID NO: 37)  5. FVETLARSICEKLEQSGL,(SEQ ID NO: 38)  6. RMFLAMITYITRNQP, (SEQ ID NO: 39) 7. LSIAPIMFSNKMARLGKGYMFESKSMKLRTQIPAEMLA, (SEQ ID NO: 40) 8. SPGMMMGMFNMLSTVLGVS, (SEQ ID NO: 41)  9. GINMSKKKSYIN,(SEQ ID NO: 42) 10. TGTFEFTSFFYRYGFVANFSMELPSFGVSGINESADMSI,(SEQ ID NO: 43) 11. GVTVIKNNMINNDLGPATAQMALQLFIKDYRYTYRCHRGDTQIQTRRSFE,(SEQ ID NO: 44) 12. VSDGGPNLY, (SEQ ID NO: 45) 13. MEYDAVATTHSW,(SEQ ID NO: 46) 14. PKRNRSILNTSQRGILEDEQMYQ, or (SEQ ID NO: 47)15. AEIMKICST;a CTL epitope of the PB2 gene has a sequence consisting of:

(SEQ ID NO: 48)  1. LMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNP, (SEQ ID NO: 49) 2. WMMAMKYPI, (SEQ ID NO: 50)  3. PERNEQGQTLWSK, (SEQ ID NO: 51) 4. PLAVTWWNRNGP, (SEQ ID NO: 52)  5. GPVHFRNQVKIRR, (SEQ ID NO: 53) 6. YIEVLHLTQGTCW, (SEQ ID NO: 54)  7. EQMYTPGGEV, (SEQ ID NO: 55) 8. NDDVDQSLIIAARNIVRRA, (SEQ ID NO: 56)  9. ASLLEMCHSTQIGG,(SEQ ID NO: 57) 10. SFSFGGFTFK, (SEQ ID NO: 58) 11. LTGNLQTLK,(SEQ ID NO: 59) 12. RVHEGYEEFTMVG, (SEQ ID NO: 60) 13. RATAILRKATRR,(SEQ ID NO: 61) 14. VAMVFSQEDCM, (SEQ ID NO: 62) 15. KAVRGDLNF,(SEQ ID NO: 63) 16. VNRANQRLNPMHQLLRHFQKDAKVLF, (SEQ ID NO: 64)17. RVSKMGVDEYS, (SEQ ID NO: 65) 18. GNVLLSPEEVSETQG, (SEQ ID NO: 66)19. LTITYSSSMMWEINGPESVL, (SEQ ID NO: 67) 20. NTYQWIIRNWE,(SEQ ID NO: 68) 21. MLYNKMEFEPFQSLVPKA, (SEQ ID NO: 69)22. LGTFDTVQIIKLLPFAAAPP, (SEQ ID NO: 70)23. QSRMQFSSLTVNVRGSGMRILVRGNSPVFNYN, or (SEQ ID NO: 71)24. GVESAVLRGFLI; anda CTL epitope of the M1 gene has a sequence consisting of:

(SEQ ID NO: 72) 1. SLLTEVETYVL, (SEQ ID NO: 73) 2. KTRPILSPLTKGIL,(SEQ ID NO: 74) 3. GFVFTLTVPSE, (SEQ ID NO: 75) 4. LYRKLKREITF,(SEQ ID NO: 76) 5. ALASCMGLIY, (SEQ ID NO: 77) 6. MGTVTTEVAFGLVCA,(SEQ ID NO: 78) 7. NRMVLASTTAKAMEQMAGSS, or (SEQ ID NO: 79)8. QARQMVQAMR.

Therefore, the NP (SEQ ID NO: 1), PA (SEQ ID NO: 3), PB1 (SEQ ID NO: 5),PB2 (SEQ ID NO: 7), and M1 (SEQ ID NO: 9) genes each are characterizedby containing at least one CTL epitope.

Preferably, a T-cell vaccine delivery vector may include a recombinantvirus, and the recombinant virus is selected from recombinant vacciniavirus, recombinant adenovirus, recombinant adeno associated virus,recombinant retrovirus, and recombinant lentivirus. More preferably, therecombinant (replicating) vaccinia virus (hereinafter, referred to as‘rVV’) may be used.

The administration route of the vaccine according to the presentinvention may be a skin scarification (s.s.), intramuscular (i.m.),intranasal (i.n.), intradermal (i.d.), intravenous (i.v.), orintraperitoneal (i.p.) route, but is not limited thereto. Anadministration method using the skin scarification (s.s.) route is mostpreferable.

The T-cell based universal influenza vaccine of the present inventionmay include at least one additive selected from pharmaceuticallyacceptable immunopotentiator, carrier, excipient, and diluent, and maybe provided in a unit-dose container or a multi-dose container, forexample, sealed ample, bottle, or the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows graph depicting T-cell immunogenicity according to theconcentration of rVV-NP vaccine administered to C57BL/6 mice by skinscarification. rVV-EGFP as a negative control was administered by skinscarification, and x-31 as a positive control was intranasallyadministered. The splenocytes stimulated with NP₃₆₆ CTL epitope peptidesafter 4 weeks of immunization were analyzed by IFN-γ-ELISPOT assay (eachbar indicates average±standard deviation (SD) for 2 mice/group.

FIG. 2 shows a graph depicting the effect according to theadministration method of rVV-NP immunization by using IFN-γ-ELISPOTactivity.

10⁶ pfu of rVV-NP vaccine was administered to the C57BL/6 mice throughthe intramuscular (i.m), intranasal (i.n.), or skin scarification (s.s.)route. The splenocytes stimulated with NP₃₆₆ CTL epitope peptides after10 days or 4 weeks of immunization were analyzed by IFN-γ-ELISPOT assay(each bar indicates average±standard deviation (SD) for 2 mice/group.

FIG. 3 shows a graph depicting the effect according to the vaccinationadministration method of x-31 by using IFN-γ-ELISPOT activity. C57BL/6mice were vaccinated with 10⁴ pfu of x-31 through the combination ofintramuscular (i.m) and intranasal (i.n.) routes two times at aninterval of 4 weeks, without anesthesia. The splenocytes stimulated withby NP₃₆₆ CTL epitope peptides after 10 days of vaccination were analyzedby IFN-γ-ELISPOT assay (each bar indicates average±standard deviation(SD) for 2 mice/group.

FIG. 4A shows a graph depicting the influenza-specific T-cell responseinduced by rVV-flu vaccination route in x-31-prime-immunized mice.x-31-primed C57BL/6 mice were challenged with 10⁶ pfu of vacciniainfluenza. rVV-flu (5) is vaccine consisting of rVV-NP, -PA, -M1, -PB2,and -PB1, and parental vaccinia (VV) or x-31 is a control. FIG. 4A showsevaluation of IFN-γ-ELISPOT activity in splenocytes stimulated withNP₃₆₆ or PA₂₂₄ epitope or PR8-infected EL4 cells after 3 weeks ofimmunization in C57BL/6 mice.

FIG. 4B shows a graph depicting the influenza-specific T-cell responseinduced by rVV-flu vaccination route in x-31-prime-immunized mice.x-31-primed C57BL/6 mice were challenged with 10⁶ pfu of vacciniainfluenza. rVV-flu (5) is vaccine consisting of rVV-NP, -PA, -M1, -PB2,and -PB1, and parental vaccinia (VV) or x-31 is a control. FIG. 4B showsevaluation of IFN-γ-ELISPOT activity in lung cells stimulated with NP₃₆₆or PA₂₂₄ epitope or PR8-infected EL4 cells after 3 weeks of immunizationin C57BL/6 mice.

FIG. 4C shows a graph depicting the influenza-specific T-cell responseinduced by rVV-flu vaccination route in x-31-prime-immunized mice.x-31-primed C57BL/6 mice were challenged with 10⁶ pfu of vacciniainfluenza. rVV-flu (5) is vaccine consisting of rVV-NP, -PA, -M1, -PB2,and -PB1, and parental vaccinia (VV) or x-31 is a control. FIG. 4C showsfunctional lytic activity by in vivo CTL assay, on day 7 afterimmunization (each bar indicates average±standard deviation (SD) for 2˜3mice/group).

FIG. 4D shows a graph depicting the influenza-specific T-cell responseinduced by rVV-flu vaccination route in x-31-prime-immunized mice.Balb/c mice were challenged with 10⁶ pfu of vaccinia influenza. rVV-flu(5) is vaccine consisting of rVV-NP, -PA, -M1, -PB2, and -PB1, andparental vaccinia (VV) or x-31 is a control. FIG. 4D shows evaluationo_(f I)FN-γ-E_(LIS)POT activity in splenocytes stimulated with NP₁₄₇epitope after 3 weeks of immunization in Balb/c mice.

FIG. 4E shows a graph depicting the influenza-specific T-cell responseinduced by rVV-flu vaccination route in x-31-prime-immunized mice.Balb/c mice were challenged with 10⁶ pfu of vaccinia influenza. rVV-flu(5) is vaccine consisting of rVV-NP, -PA, -M1, -PB2, and -PB1, andparental vaccinia (VV) or x-31 is a control. FIG. 4E shows ev^(a)luationof IFN-γ-ELISPOT activity in lung cells stimulated with NP₁₄₇ epitopeafter 3 weeks of immunization in Balb/c mice.

FIG. 4F shows a graph depicting the influenza-specific T-cell responseinduced by rVV-flu vaccination route in x-31-prime-immunized mice.Balb/c mice were challenged with 10⁶ pfu of vaccinia influenza. rVV-flu(5) is vaccine consisting of rVV-NP, -PA, -M1, -PB2, and -PB1, andparental vaccinia (VV) or x-31 is a control. FIG. 4F shows functionallytic activity by in vivo CTL assay, on day 7 after immunization (eachbar indicates average±standard deviation (SD) for 2˜3 mice/group).

FIG. 5A shows a graph depicting secretion of multifunctional cytoki_(ne)from influenza-specific T-cells. The splenocytes of C57BL/6 mice wereused, and stimulated with NP₃₆₆ (upper panel) or PA₂₂₄ (lower panel)epitopes after 7 weeks of rVV-flu immunization. Secretion of IFN-γ,TNF-α and IL-2 cytokines and expression of degranulation marker (CD107a)were measured by in vivo cytokine staining: FIG. 5A shows the rate (%)of CD8 T-cells secreted by each cytokine.

FIG. 5B shows a graph depicting secretion of multifunctional cytoki_(ne)from influenza-specific T-cells. The splenocytes of C57BL/6 mice wereused, and stimulated with NP₃₆₆ (upper panel) or PA₂₂₄ (lower panel)epitopes after 7 weeks of rVV-flu immunization. Secretion of IFN-γ,TNF-α and IL-2 cytokines and expression of degranulation marker (CD107a)were measured by in vivo cytokine staining: FIG. 5B shows measurementresults of T-cell response and degranulation for various cytokinecombina_(tio)ns in order to anal_(yze) multi-functionality of theinfluenza-specific T-cells (each bar indicates average±standarddeviation (SD) for 2 mice/group).

FIG. 5C shows a graph depicting secretion of multifunctional cytokinefrom influenza-specific T-cells. The splenocytes of C57BL/6 mice wereused, and stimulated with NP₃₆₆ (upper panel) or PA₂₂₄ (lower panel)epitopes after 7 weeks of rVV-flu immunization. Secretion of IFN-γ,TNF-α and IL-2 cytokines and expression of degranulation marker (CD107a)were measured by in vivo cytokine staining: FIG. 5C shows dot plots fora positive (+) control and rVV-NP+PA.

FIG. 6 shows a graph depicting weight loss for 7 days when C57BL/6 micewere challenged with PR8 (0.5/1_(0/1)0_(0 L)D₅₀), after immunization(each bar indicates average±standard error of measurement (SEM) for 4mice/group).

FIG. 7A shows graph depicting weight loss and survival rate whenx-31-prime-immunized C57BL/6 mice were challenged with PR8 (100 LD₅₀),after 3 weeks of rVV-flu immunization. The % weight loss were trackedfor 2 weeks (each bar indicates average±standard error of measurement(SEM) for 8 mice/group.

FIG. 7B shows graph depicting weight loss and survival rate whenx-31-prime-immunized Balb/mice were challenged with PR8 (100 LD₅₀),after 3 weeks of rVV-flu immunization. The % weight loss were trackedfor 2 weeks (each bar indicates average±standard error of measurement(SEM) for 8 mice/group).

FIG. 7C shows a graph depicting weight loss and survival rate whenx-31-prime-immunized C57BL/6 mice were challenged with PR8 (100 LD₅₀),after 3 weeks of rVV-flu immunization. The % survival were tracked for 2weeks (each bar indicates average±standard error of measurement (SEM)for 8 mice/group.

FIG. 7D shows a graph depicting weight loss and survival rate whenx-31-prime-immunized Balb/c mice were challenged with PR8 (100 LD₅₀),after 3 weeks of rVV-flu immunization. The % survival were tracked for 2weeks (each bar indicates average±standard error of measurement (SEM)for 8 mice/group).

FIG. 7E shows a graph depicting weight loss and survival rate whenx-31-prime-immunized C57BL/6 and Balb/c mice were challenged with PR8(100 LD₅₀), after 3 weeks of rVV-flu immunization. Lung virus titerswere measured on day 3, 5, and 7 post challenge (each bar indicatesaverage±standard error of measurement (SEM) for 3 mice/group).

FIG. 7F shows a graph depicting weight loss and survival rate whenx-31-prime-immunized C57BL/6 and Balb/c mice were challenged with PR8(100 LD₅₀), after 3 weeks of rVV-flu immunization. Lung virus titerswere measured on day 3, 5, and 7 post challenge (each bar indicatesaverage±standard error of measurement (SEM) for 3 mice/group).

FIG. 8A shows a graph depicting the weight loss and survival rate ofx-31-prime-immunized C57BL/6 mice, which were challenged with HPAI H5N1after 3 weeks of rVV-flu immunization (100 LD₅₀ A/MD/W401/11 (HPAI H5N1field isolate)). The % weight loss were tracked for 2 weeks (each barindicates average±standard error of measurement (SEM) for 8 mice/group).

FIG. 8B shows graph depicting the weight loss and survival rate ofx-31-prime-immunized C57BL/6 mice, which were challenged with HPAI H5N1after 3 weeks of rVV-flu immunization (100 LD₅₀ A/MD/W401/11 (HPAI H5N1field isolate)). The % survival were tracked for 2 weeks (each barindicates average±standard error of measurement (SEM) for 8 mice/group).

FIG. 8C shows a graph depicting the weight loss and survival rate ofx-31-prime-immunized C57BL/6 mice, which were challenged with HPAI H5N1after 3 weeks of rVV-flu immunization (100 LD₅₀ A/MD/W401/11 (HPAI H5N1field isolate)). Lung virus titers were measured on day 3, 5, and 7(each bar represents average±standard error of measurement (SEM) for 3mice/group).

FIG. 9A shows a graph depicting cross-reactive IFN-γ-ELISPOT activitycorresponding to CTL epitope, after the prime-immunized Phil/2/82 micewere subjected to rVV-flu immunization. The cross-reactive IFN-γ-ELISPOTactivity was measured on CTL epitope of the priming virus (Phil/2/82)and the challenging virus (Cal/04/09). The above assay was conductedafter 4 weeks of priming. (each bar indicates average±standard deviation(SD) for 2 mice/group).

FIG. 9B shows a graph depicting cross-reactive IFN-γ-ELISPOT activitycorresponding to CTL epitope, after the prime-immunized Phil/2/82 micewere subjected to rVV-flu immunization. The cross-reactive IFN-γ-ELISPOTactivity was measured on CTL epitope of the priming virus (Phil/2/82)and the challenging virus (Cal/04/09). The above assay was conductedafter 3 weeks of rVV-flu vaccination (each bar indicatesaverage±standard deviation (SD) for 2 mice/group).

FIG. 10A shows a graph depicting the weight loss and survival rate ofPhil/2/82-prime-immunized C57BL/6 mice, which were challenged withpandemic H1N1 adapted into mice after rVV-flu immunization. The micewere challenged with Cal/04/09 virus adapted into 100 LD₅₀ mice, after 3weeks of immunization, and the % weight loss were observed (each barindicates average±standard error of measurement (SEM) for 8 mice/group).

FIG. 10B shows a graph depicting the weight loss and survival rate ofPhil/2/82-prime-immunized C57BL/6 mice, which were challenged withpandemic H1N1 adapted into mice after rVV-flu immunization. The micewere challenged with Cal/04/09 virus adapted into 100 LD₅₀ mice, after 3weeks of immunization, and the % survival thereof were observed (eachbar indicates average±standard error of measurement (SEM) for 8mice/group).

DETAILED DESCRIPTION OF EMBODIMENTS

Hereinafter, the present invention will be described in detail withreference to examples and drawings. The examples are only forspecifically explaining the present invention and it is obvious to thoseskilled in the art that the scope of the present invention is notlimited by the examples according the gist of the present invention.

Preparation of Animal Model and Virus

C57BL/6 and Balb/c female mice of 6 weeks of age purchased from theCharles-River Laboratory (Orient Bio Inc., Sungnam, Korea) were used inthe present invention. Parental NYCBH vaccinia variants were used forproduction of rVV-flu, and A/PR/8/34 (H1N1) and x-31 (H3N2) influenzaviruses were used in the present invention. All procedures for using theH5N1 virus were conducted in a Biosafety Level-3 (BSL-3) facility.

Selection of Internal Genes Containing Consensus Sequence andRepresentative Sequence

In order to determine the equidistant consensus sequence from bird, pig,and human influenza isolates, a total of 14,011 sequences of theinfluenza internal genes (PB2, PB1, PA, NP, M1, M2, NS1 and NS2) weresearched from influenza virus data of the U.S. National Center forBiotechnology Information (NCBI)(http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).

In respective species, the individual gene phylogenetic tree was createdby using a neighbor-joining method, and, finally, the consensus sequenceof eight kinds of internal genes was determined. Three among the eightkinds of internal genes, NS1, NS2, and M2, were excluded in selection ofthe internal genes for the T-cell based universal flu vaccine of thepresent invention due to the following reasons:

-   (1) Since three kinds of internal genes are shorter than the other    internal genes, the opportunities for presenting a defense T-cell    epitope are limited;-   (2) Since NS1 has been known to suppress Type I interferon response,    it may influence the immunogenicity formation when a delivery vector    using NS1 as an antigen is used; and-   (3) M2 does not contribute to the defense against a fatal dose of    vaccination since the efficacy caused by the combination of    rVV-NP+PA and rVV-flu (5) is significantly lower than the efficacy    induced by single r-VV-M2 vaccination in an anti-M2 antibody    response.

Therefore, five kinds of internal genes, NP, PA, PB1, PB2, and M1 wereselected for the T-cell based universal flu vaccine of the presentinvention, and immunogenicity and efficacy thereof were analyzed.

GenBank Accession Numbers of the five kinds of internal genes andproteins translated therefrom are as follows:

-   NP gene (SEQ ID NO: 1), gi:323984, gb:M22574.1,-   NP protein (SEQ ID NO: 2), gi:323985, gb:AAA43095.1;-   PA gene (SEQ ID NO: 3), gi:78097605, gb:CY005610.1,-   PA protein (SEQ ID NO: 4), gi:78097606, gb:ABB20301.1;-   PB1 gene (SEQ ID NO: 5), gi:82654856, gb:CY005794.1,-   PB1 protein (SEQ ID NO: 6), gi:82654857, gb:ABB88367.1;-   PB2 gene (SEQ ID NO: 7), gi:134044357, gb:CY011035.2,-   PB2 protein (SEQ ID NO: 8), gi:113901268, gb:ABI47980.1; and-   M1 gene (SEQ ID NO: 9), gi:4584881, gb:AAD25173.1,-   M1 protein (SEQ ID NO: 10), gi:4584882, gb:AAD25173.1,

TABLE 1 Numbers of influenza isolates and subgroups for determiningfinal consensus sequences of influenza A internal genes. Number ofsubgroups Final consensus Number of influenza (selection criteria)sequence isolates analyzed (% Ave.a.a.identy ± SD) Average Human PigBird Total Human Pig Bird Total Distance PB2 866 187 1303 2356 32 (>97%)13 36 81 97.70% −96% −97% PB1 835 199 1213 2247 30 (>97%)  7 (>91%) 45(>98%) 82 98.70% PA 826 190 1403 2419 18 (>97%) 12 (>94%) 31 (>96%) 61 9790% NP 581 187 920 1688 29 (>97%) 27 (>96%) 55 (>96%) 111 96.80% M1255 130 525 910 19 (>97%) 18 (>97%) 28 65 97.70% −96% M2 372 137 5361045 32 (>91%) 32 (>93%) 29 (>91%) 93 92.50% (95.0% ± 2.4%) (95.5% ±1.5%) 94.7% ± 1.8%) NS1 710 228 1419 2357 30 (>94%) 32 (>89%) 76 (>90%)138 90.80% (95.8% ± 2.6%) (96.5% ± 2.2%) NS2 256 111 622 989 20 (>97%)15(>91%) 19(>91%) 54 94.40% Total 14,011 685 97.10%

The T-cell based universal flu vaccine containing the five kinds ofinternal genes has higher immunogenicity than the control group. Whencomparing the conventionally known conserved human T-cell epitope withthe consensus sequence from the influenza isolates, 1,142 amino acidsamong 1,147 amino acids of the conserved human T-cell epitope are 99.6%identical to the consensus sequence.

TABLE 2 Homology between known common epitope and consensus sequence ofT-cell based universal flu vaccine of the present invention Total numberof amino Number of amino acids of acids of epitope homologous epitopeHomology % PB2 366 365 99.7% PB1 359 358 99.7% PA 171 170 99.4% NP 149148 99.3% M1 102 101 99.0% Total 1147. 1142 99.6%

The internal genes of the present invention have average 95% sequencehomology to the new influenza (2009 H1N1) (95.3% toA/Mexico/InDRE4114/2009; 95.2% to A/Canada-AB/RV1532/2009; and 95.0% toA/New York/1669/2009), and average 97.8% amino acid homology to theinfluenza isolates from influenza virus data of the U.S. National Centerfor Biotechnology Information (NCBI). The above homology level meansthat the present invention can achieve broad T-cell-mediated defenseagainst some unknown epidemic influenza.

TABLE 3 Homology between influenza isolates from influenza virus data ofthe U.S. National Center for Biotechnology Information (NCBI) andconsensus sequence of T-cell based universal flu vaccine of the presentinvention % Homology of influenza variant having consensus sequence (%range of Number influenza isolates in influenza database)* of Mallard/Philippinne/ California/ amino 100% Average PR/8/34 W401/11 2/82 04/09acids identical distance (H1N1) (H5N1) (H3N2) (H1N1) PB2 759A/blue-winged 97.7% 97.2% 98.4% 95.5% 97.8% Teal/Ohio/926/ (63.5%)(57.8%) (75.6%) (59.3%) 2002 (H3N8) PB1 758 A/turkey/Italy/4169/ 98.7%97.6% 98.5% 98.8% 97.1% 1999 (H7N1) (87.5%) (63.9%) (63.3%) (87.9%) PA716 A/chicken/ 97.9% 97.2% 99.2% 95.4% 97.5% Hong Kong/ (65.2%) (38.1%)(81.2%) (63.5%) 17/1977 (H6N1) NP 498 A/duck/Bavaria/2/ 96.8% 94.6%98.0% 92.4% 94.6% 1977 (H1N1) (69.3%) (55.0%) (86.4%) (69.3%) M1 257A/chicken/ 97.7% 96.8% 96.4% 97.2% 96.5% New York/ (67.3%) (86.0%)(57.6%) (67.3%) 13142-5/94 (H7N2N5B) Total 2983 100.0% 97.8% 96.7% 98.1%95.9% 95.9% number of (71.3%) (56.5%) (74.1%) (69.9%) Amino acids *Range(%) of influenza isolates in influenza virus data of the U.S. NationalCenter for Biotechnology Information (NCBI), which share higher homologythan variant appeared in the consensus sequence: PB2 (n = 2356), PB1 (n= 2247), PA (n = 2419), NP (n = 1688), M1 (n = 910).

EXAMPLE 1 Production of rVV-flu Vaccine

In order to produce r-VV flu vaccine, influenza gene-optimized humancodons were synthesized, and inserted at the EcoRV site of pUc57(provided by GenScript (Piscataway, N.J.). Each plasmid containing NP orMI gene was cut by AscI/SbfI restriction enzymes, and then the cutpBMSF7C vaccine delivery vector was linked thereto by PstI/AscI. Anappropriate size of PB2, PB1, and PA were inserted into the cutfragments of pUC57 by AscI/SbfI, and then the vector was cut by DraIrestriction enzyme one more time. The cut pBMSF7C was cloned byPstI/AscI. The rVV-flu vaccine was produced by slightly changing theconventional method thereof (Vaccine 25,630-637, 2007). BHK-21 cellsinfected with vaccinia virus at multiplicitiy of infection (MOI) of 10were transfected with pBMSF7C-flu. The viruses were harvested after 24hours of transfection, and RK-13 cells, from which recombinant HAnegative plaques are to be screened, were again infected therewith.After 3 days of infection, the cells were reacted with turkey RBC at 37°C. for 30 minutes to isolate HA negative plaques. The HA negativeplaques were continuously cloned until HA positive plaques disappeared,and then confirmed by HA staining of ˜1,000 plaques per 100-mm dish. Thefinal recombinant viruses were purified, and amplified in BHK-21 cells(0.1 MOI). They were low-temperature stored at −80° C. prior to the usethereof.

It was confirmed whether the influenza genes are inserted into therecombinant vaccinia virus by using the PCR method, and the final cloneswere confirmed by Western blot and sequencing of binding sites.

EXAMPLE 1-1 Western Blot Assay of rVV-flu

BHK-21 cells were infected with rVV-flu at MOI of 0.1 for 1 hour. Then,the cells were incubated for three days, followed by harvesting.Proteins were extracted with an M-PER buffer solution according to theconventionally known existing method (Pierce, Rockford, Ill.).Thermo-denatured proteins were separated by 4-12% SDS-PAGE (InvitrogenCarlsbad, Calif.), and transferred on the nitrocellulose membrane(Invitrogen Carlsbad, Calif.). The membrane was blocked in a PBS buffersolution containing 0.2% Tween 20 together with 5% nonfat milk for 1hour, and reacted with primary antibodies.

The following antibodies were used for detection:

-   anti-PB2 (Santacruz Carlsbad, Calif.);-   anti-PB1 (Santacruz Carlsbad, Calif.);-   anti-PA (Peptron Inc., Daejeon, Korea);-   anti-NP and anti-M1 (X-31/PR8 immunized mouse serum).

The membrane was incubated together with horseradish peroxidase-labeledanti-mouse IgG antibodies (KPL Gaithersburg, Md.), and stained with ECLplus kit (Amersham Buckinghamshire, U.K.).

EXAMPLE 2 Immunogenicity Evaluation of rVV-flu EXAMPLE 2-1 IFN-γ ELISPOTAssay

In the present invention, immunogenicity of rVV-flu was evaluatedthrough IFN-γ ELISPOT assay in order to determine the optimum vaccinecombination in vaccinia based influenza vaccination. High immunogenicityof nucleoprotein (NP) was confirmed in the H-2b mouse model. Severaldoses of rVV-NP were administered through the conventionally known skinscarification (s.s.), and immunogenicity due to this was inducedaccording to the administration dose In T-cell response. It wasconfirmed that the boosting immunization improved the T-cell response(see, FIG. 1).

In the present invention, the administration of vaccine was performed byan intramuscular (i.m.) method, an intranasal (i.n.) method, or a skinscarification (s.s.) method. Here, the skin scarification (s.s.) methodwas most preferable since it exhibits 5 times higher immunogenicity thanthe intramuscular (i.m.) method or the intranasal (i.n.) method (see,FIG. 2).

Since 91˜98% of humans have been infected with influenza virus, theyhave influenza-specific memory T-cells. Therefore, in the presentinvention, influenza-prime-immunized mice were used as a mouse model forevaluating immunogenicity and efficacy of vaccine. Among fourcombinations of the intramuscular (i.m.) method and the intranasal(i.n.) method, x-31 intramuscular priming/intranasal boostingadministration method induced the highest level of T-cell immunity (see,FIG. 3).

The x-31-prime-immunized C57BL/6 mice were challenged with 10⁶ pfu ofvaccinia influenza, and the Balb/c mice were challenged with 10⁶ pfu ofvaccinia influenza.

In the C57BL/6 mice, immunogenically predominant epitopes of thefollowing two kinds of T-cell based universal flu vaccine were NP₃₆₆ andPA₂₂₄, and 90% or more of flu-specific T-cell response could be induced(see, FIGS. 4A to 4F).

-   rVV-NP+PA-   rVV-flu (5) (Flu (5) means internal genes, NP, PA, PB1, PB2, and    M1.)

The epitopes of NP and PA, NP₃₆₆ and PA₂₂₄, exhibited the mostpredominant immunogenicity in flu-specific T-cell response. However,vaccine containing T-cell epitopes obtained from the five kinds ofinternal genes is provided, to thereby allow human to prepare for a casewhere NP and PA are contained as T-cell epitope.

EXAMPLE 3 Immunization Using Mouse Model

5 ul of 10⁴ pfu of X-31 was administered to the mice through theintranasal (i.n.) route without anesthesia (priming immunization, on day0). The mice were vaccinated with 5 ul of 10⁶ pfu of rVV-flu by skinscarification (s.s.) administration (boosting immunization, on day 28).

The parental-VV was used alone as a negative control. The primingvaccination of a positive control of 50 ul was performed on the thighmuscle at the rear parts of both hind legs through the intramuscular(i.m.) route, and the boosting vaccination was performed by using theintranasal (i.n.) route (10⁴ pfu of x-31).

Eight mice were used for each group. On the day after three weeks of theimmunization, the mice were anesthetized with avertin, and infected with50 ul of A/PR/8/34 virus at LD₅₀ of 0.5/10/100.

As the hetero-subtypic flu virus, 100 LD₅₀ highly pathogenic H5N1A/Mallard Duck/Korea/W401/11 and epidemic A/California/04/09 were used.The survival and the change in weight of the mice were confirmedeveryday for 2 weeks, and the mice were euthanized when they lost 30% ormore of the initial weight for animal welfare (see, FIGS. 6, 7A to 7F,10A and 10B).

EXAMPLE 4 Preparation of Lung Cells for Cell Immunity

The lung cells were disrupted following the conventionally knownprotocol (Miltenyi Biotec Inc., Carlsbad, Calif.). The disrupted lungcells were treated with RBC lysis buffer on ice for 5 minutes(Sigma-Aldrich). The lung cells with the removal of RBC weredisentangled in RPMI, and in vivo apoptosis effect (in vivo CTL) assayand ELISPOT (Enzyme-Linked ImmunoSpot) assay were conducted.

EXAMPLE 5 Ex-Vivo IFN-γ ELISPOT Assay

The ELISPOT assay was conducted using the mouse IFN-γ ELISPOT^(PLUS) kit(Mabtech, AB Nacta Strand, Sweden). 2×10⁵ fresh splenocytes or lungcells were plated on the 96-well plate coated with anti-mouse IFN-γcapture antibody, and stimulated with 1 μM of influenza peptide epitope(Peptron, Daejeon, Korea) or EL4 (2×10⁵ cells/well) infected with 1 MOIof PR8 virus for 18 hours.

The flu-specific CD8+ T-cell epitope was as follows:

NP₃₆₆₋₇₄ (Db, ASNENMETM); PA₂₂₄₋₃₃ (Db, SSLENFRAYV); PB2₁₉₆₋₂₁₀,(Db or Kb, CKIAPLMVAYMLERE); PB1₇₀₃₋₁₁ (Kb, SSYRRPVGI); and M1₁₂₈₋₃₅(Kb, MGLIYNRM).

The splenocytes pulsed with PB2₁₉₈₋₂₀₆ or infected withB/Malaysia/2506/04 were used in a level of 8±5(SD) and 13±9(SD) per wellfor a negative control. The lung cells were used in a higher level for ahigher negative control (14±13 and 25±26). The number of IFN-γ secretioncells ((ISCs) was measured using an ELISPOT reader (AID, Strassberg,Germany) (See, FIGS. 5A to 5C).

EXAMPLE 6 In Vivo Cytotoxic T-Cell (CTL) Assay

The splenocytes obtained from naive mice were stained with 5 M PKH26(Sigma-Aldrich, Saint Louis, Mo.) at room temperature for 10 minutes.Labeling was stopped by addition of the same amount of FBS, followed byincubation for 1 minute. Additional staining with 0.5 uM, 2 uM, or 8 uMof CFSE (Carboxy Fluorescein Succinimidyl Ester) was conducted at 37° C.for 10 minutes, and then stopped by addition of the same amount of FBS,followed by incubation for 1 minute. The doubly stained cells werestimulated with the indicator epitope peptide (1 uM) at 37° C. for 1hour.

Each target cell and a different peptide were mixed together, and themixture was administered to the naive or immunized mice throughintravenous (i.v.) administration. After 12 hours of administration,single cell dispersions of the splenocytes and lung cells were prepared,and activity of in vivo cytotoxic T cell (CTL) was measured using flowcytometry analysis (BD LSRFortessa, Bioscience BD, Piscataway, N.J.).The cell killing ratio (%) was calculated according to the followingequation.

$\begin{matrix}{{{Cell}\mspace{14mu}{killing}\mspace{14mu}{ratio}\mspace{14mu}(\%)} = {100 - \begin{Bmatrix}\left( {{number}\mspace{14mu}{of}\mspace{14mu}{stimulated}\mspace{14mu}{target}\mspace{14mu}{cells}\mspace{14mu}{remaining}} \right. \\{{in}{\mspace{11mu}\;}{the}\mspace{14mu}{immunized}\mspace{14mu}{{mouse}/{number}}\mspace{14mu}{of}} \\{{{un}\text{-}{stimulated}\mspace{14mu}{target}\mspace{14mu}{cells}\mspace{14mu}{remaining}\mspace{14mu}{in}\mspace{14mu}{the}}\mspace{14mu}} \\{\left. {{immunized}\mspace{14mu}{mouse}} \right)/\left( {{number}\mspace{14mu}{of}\mspace{14mu}{stimulated}} \right.} \\{{target}\mspace{14mu}{cells}\mspace{14mu}{remaining}\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{naïve}\mspace{14mu}{{mouse}/{number}}} \\{{of}\mspace{14mu}{un}\text{-}{stimulated}\mspace{14mu}{target}\mspace{14mu}{cells}\mspace{14mu}{remaining}\mspace{14mu}{in}} \\{\left. {{the}\mspace{14mu}{naïve}\mspace{14mu}{mouse}} \right) \times 100}\end{Bmatrix}}} & \lbrack{Equation}\rbrack\end{matrix}$

EXAMPLE 7 In Vivo Cytokine Staining (ICS) and T Cell PolyfunctionalityAnalysis

In the present example, polyfunctionality of influenza specific T cellinduced by rVV-flu vaccine was observed. After rVV-flue immunization,stimulations with NP₃₆₆(upper panel) and PA₂₂₄ (lower panel) epitopeswere conducted for 7 days. Secretion of IFN-γ, TNF-α and IL-2 cytokinesand expression of degranulation markers (CD107a) were measured by invivo cytokine staining (see, FIGS. 5A to 5C).

In the presence of anti-CD107a-PE-Cy7 (BD Biosciences), the splenocyteswere stimulated with cytotoxic T cell (CTL) epitope peptide (1 ug/ml)such as NP₃₆₆ or PA₂₂₄, and, after 1 hour, monesin (GolgiStop, BDBiosciences), which is a kind of transferring antibiotic materials forimproving cell permeability, was added. After 5 hours of incubation, thesplenocytes were stained with ethidium monoazide, and anti-CD3-HorizonV500, anti-CD4-Horizon V450, and anti-CD8-APC-H7 (BD Biosciences) werestained with ethidium monoazide. They were permeabilized usingCytofix/Cytoperm kit (BD Biosciences) and further stained withanti-IL-2-FITC (eBioscience), anti-IFN-γ-APC (BD Biosciences) andanti-TNF-PE (BD Biosciences). FACS assay was conducted using an LSRIIflow cytometer (BD Biosciences), and data were analyzed by FlowJosoftware (Treestar, San Fran Carlos, Calif.). Quantification andanalysis of T cells with respect to various combinations of cytokinesand degranulation were conducted by using FlowJo software.

Influenza HA is a main target for monitoring antibody-mediated hostimmunity, and variation thereof continuously occurs. Therefore, onlyinfluenza HA having a consensus sequence has an advantage ofcross-defense. The internal genes have relatively low immunity than HA,and thus have been significantly conserved. The conserved sequence ofthe present invention is characterized by having sufficient cellimmunity for allowing the cross-reaction since it has average 97.8%homology The present invention is characterized by selecting the entireinternal genes in their original states, instead of epitope sequences ofproviding customized antigen representation of T cell epitope accordingto individual HLA polymorphism. In addition, the multi-epitope-specificT cell response can decrease the possibility of variant formation.Notwithstanding the use of the multi-gene, the decrease inimmunogenicity of individual antigen is due to antigen competition amongindividual recombinant viruses in replication of T cells and antigenpresentation. This was demonstrated from antigen competition in the DNAvaccine combination study. In the present invention, cross-reactiveIFN-γ-ELISPOT activity was measured with respect to CTL epitopes of thepriming virus (Phil/2/82) and the challenging virus (Cal/04/09). Theassay was conducted after 4 weeks of priming (a) and after 3 weeks ofrVV-flu vaccination (see, FIGS. 9A and 9B).

EXAMPLE 8 Titer Assay of Lung Virus

The mice infected with influenza were enthanized after 3, 5, and 7 days,to obtain lung tissues thereof. The obtained lung tissues were disruptedin 10% W/V serum free Minimum Essential Me-dia (MEM) for analyzing virustiter using plaque assay. The limit of virus detection was 100 pfu/g ofthe lung (this value corresponds ˜20 pfu/mouse). The titer of HPAI H5N1virus was measured by using the eggs in BSL3+ facilities receivedapproval for influenza experiment. The limit of virus detection was 0.7log 10 EID₅₀/ml or lower (see, FIGS. 7A to 7F, and 8A to 8C).

The differences in weight loss and lung virus titer between groups wereanalyzed by the Student T test. The difference in survival wascalculated by the Fisher's exact test, and it was confirmed that thestatically significant p value was 0.05 or lower (see, FIGS. 7A to 7F,and 8A to 8C).

The T cell-based universal influenza vaccine of the present inventionincludes at least one CTL epitope in each of the internal genes, andthus can enhance immunogenicity thereof according to combination of theinternal genes, and thereby can defend against various hetero-subtypicvariants of the influenza viruses.

What is claimed is:
 1. A T cell-based immunogenic composition comprisingan influenza M1 gene having the sequence of SEQ ID NO:9, furthercomprising at least one additive selected from the group consisting ofpharmaceutically acceptable immunopotentiator, carrier, excipient, anddiluent.
 2. The T cell-based immunogenic composition of claim 1, furthercomprising at least one influenza gene selected from the groupconsisting of an NP gene having the sequence of SEQ ID NO:1, a PA genehaving the sequence of SEQ ID NO:3, a PB1 gene having the sequencedescribed by SEQ ID NO: 5, and a PB2 gene having the sequence describedby SEQ ID NO:
 7. 3. A T cell-based immunogenic composition comprisingall of NP (SEQ ID NO: 1), PA (SEQ ID NO: 3), PB1 (SEQ ID NO: 5), PB2(SEQ ID NO: 7), and M1 (SEQ ID NO: 9) gene.
 4. The T cell-basedimmunogenic composition of claim 2, wherein the NP (SEQ ID NO: 1), PA(SEQ ID NO: 3), PB1 (SEQ ID NO: 5), PB2 (SEQ ID NO: 7), and M1 (SEQ IDNO: 9) gene each encodes at least one CTL epitope.
 5. The T cell-basedimmunogenic composition of claim 2, wherein the CTL epitope of the NPprotein is MASQGTKRSYEQMET (SEQ ID NO: 11), GIGRFYIQMCTELKL (SEQ ID NO:12), MVLSAFDERRN (SEQ ID NO: 13), YLEEHPSAGKDPKKTGGPIY (SEQ ID NO: 14),LYDKEEIRRIWRQANNG (SEQ ID NO: 15), ATYQRTRAL (SEQ ID NO: 16), YERMCNILKG(SEQ ID NO: 17), QVRESRNPGNAEIEDLIFLA (SEQ ID NO: 18),QLVWMACHSAAFEDLRVSSF (SEQ ID NO: 19), or QPTFSVQRNLPF (SEQ ID NO: 20);the CTL epitope of the PA protein is KIETNKFAAICTHLEVCFMYSDFHF (SEQ IDNO: 21), RTMAWTVVNSI (SEQ ID NO: 22), VEKPKFLPDLY (SEQ ID NO: 23),YYLEKANKIKSE (SEQ ID NO: 24), THIHIFSFTGEEMA (SEQ ID NO: 25),RGLWDSFRQSERGEETIEE (SEQ ID NO: 26), RSKFLLMDALKLSIE (SEQ ID NO: 27),HEGEGIPLYDAIKC (SEQ ID NO: 28), SQLKWALGENMA (SEQ ID NO: 29),EFNKACELTDSSWI (SEQ ID NO: 30), SRPMFLYVRTNGTSK (SEQ ID NO: 31), orAESRKLLLI (SEQ ID NO: 32); the CTL epitope of the PB1 protein isDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSE (SEQ ID NO:33),MAFLEESHPGIFENS (SEQ ID NO: 34), VQQTRVDKLTQGRQTYDWTLNRNQPAATALANTIE(SEQ ID NO: 35), TKKMVTQRTIGKKK (SEQ ID NO: 36), FVETLARSICEKLEQSGL (SEQID NO: 37), RMFLAMITYITRNQP (SEQ ID NO: 38),LSIAPIMFSNKMARLGKGYMFESKSMKLRTQIPAEMLA (SEQ ID NO: 39),SPGMMMGMFNMLSTVLGVS (SEQ ID NO: 40), GINMSKKKSYIN (SEQ ID NO: 41),TGTFEFTSFFYRYGFVANFSMELPSFGVSGINESADMSI (SEQ ID NO: 42),GVTVIKNNMINNDLGPATAQMALQLFIKDYRYTYRCHRGDTQIQTRRSFE (SEQ ID NO: 43),VSDGGPNLY (SEQ ID NO: 44), MEYDAVATTHSW (SEQ ID NO: 45),PKRNRSILNTSQRGILEDEQMYQ (SEQ ID NO: 46), or AEIMKICST (SEQ ID NO: 47);the CTL epitope of the PB2 gene is LMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNP(SEQ ID NO: 48), WMMAMKYPI (SEQ ID NO: 49), PERNEQGQTLWSK (SEQ ID NO:50), PLAVTWWNRNGP (SEQ ID NO:51), GPVHFRNQVKIRR (SEQ ID NO: 52),YIEVLHLTQGTCW (SEQ ID NO: 53), EQMYTPGGEV (SEQ ID NO: 54),NDDVDQSLIIAARNIVRRA (SEQ ID NO: 55), ASLLEMCHSTQIGG (SEQ ID NO: 56),SFSFGGFTFK (SEQ ID NO: 57), LTGNLQTLK (SEQ ID NO: 58), RVHEGYEEFTMVG(SEQ ID NO: 59), RATAILRKATRR (SEQ ID NO: 60), VAMVFSQEDCM (SEQ ID NO:61), KAVRGDLNF (SEQ ID NO: 62), VNRANQRLNPMHQLLRHFQKDAKVLF (SEQ ID NO:63), RVSKMGVDEYS (SEQ ID NO: 64), GNVLLSPEEVSETQG (SEQ ID NO: 65),LTITYSSSMMWEINGPESVL (SEQ ID NO: 66), NTYQWIIRNWE (SEQ ID NO: 67),MLYNKMEFEPFQSLVPKA (SEQ ID NO: 68), LGTFDTVQIIKLLPFAAAPP (SEQ ID NO:69), QSRMQFSSLTVNVRGSGMRILVRGNSPVFNYN (SEQ ID NO: 70), or GVESAVLRGFLI(SEQ ID NO: 71); and the CTL epitope of the M1 protein is SLLTEVETYVL(SEQ ID NO: 72), KTRPILSPLTKGIL (SEQ ID NO: 73), GFVFTLTVPSE (SEQ ID NO:74), LYRKLKREITF (SEQ ID NO: 75), ALASCMGLIY (SEQ ID NO: 76),MGTVTTEVAFGLVCA (SEQ ID NO: 77), NRMVLASTTAKAMEQMAGSS (SEQ ID NO: 78),or QARQMVQAMR (SEQ ID NO: 79).
 6. The T cell-based immunogeniccomposition of claim 2, wherein the genes are included in a recombinantvirus.
 7. The T cell-based immunogenic composition of claim 6, whereinthe recombinant virus is a recombinant vaccinia virus, a recombinantadenovirus, a recombinant adeno associated virus, a recombinantretrovirus, or a recombinant lentivirus.
 8. The T cell-based immunogeniccomposition of claim 7, wherein the recombinant virus is the recombinantvaccinia virus.
 9. The T cell-based immunogenic composition of claim 7,wherein it is administered through a skin scarification (s.s) route, anintramuscular (i.m.) route, an intranasal (i.n.) route, an intradermal(i.d.) route, an intravenous (i.v.) route, or an intraperitoneal (i.p.)route.
 10. The T cell-based immunogenic composition of claim 9, whereinit is administered through the skin scarification (s.s.) route.